Tyrosine hydroxylase purified to homogeneity from cultured rat pheochromocytoma cells is inactivated by incubation with its reduced pterin confactors L-erythro- tetrahydrobiopterin (BH4), 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin (6MPH4) and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin (DMPH4). Each of the two diastereoisomers of L-erythro-tetrahydrobiopterin inactivates tyrosine hydroxylase but the natural (6R) form is much more potent than the unnatural (6S) form at equimolar concentrations. The pterin analog 6-methyl-5-deaza-tetrahydropterin, which has no cofactor activity with tyrosine hydroxylase, also inactivates the enzyme whereas the oxidized pterina 7,8 dihydrobiopterin and biopterin do not. The inactivation process is both temperature and time dependent and results in a reduction of the Vmax for both tetrahydrobiopterin and tyrosine. Neither tyrosine nor oxygen inactivates tyrosine hydroxylase. However, incubation of the enzyme with BH4 in the absence of oxygen prevents pteridine-induced inactivation. The phosphorylated forms of tyrosine hydroxylase is also quite sensitive to inactivation by reduced pteridines.